Project Summary Mounting evidence have demonstrated proline hydroxylation (Hyp) as a fundamental posttranslational modification that are highly responsive to the changes in cellular metabolic environment. During cancer development, rapid proliferation of cancer cells in solid tumors suffers from limited oxygen supply. The hypoxia microenvironment prevents its hydroxyproline-dependent degradation of HIF? proteins and activates hypoxia- response cellular pathways that promote cancer cell survival in hypoxia. In addition to oxygen, the regulatory enzyme prolyl hydroxylases are also sensitive to the concentration of iron and key mitochondria metabolites including succinate, fumarate and alpha-ketoglutarate, making the pathway a critical metabolic sensor in cells. Extensive studies have demonstrated that proline hydroxylation of substrate proteins regulates protein-protein interactions or substrate protein degradation. Despite of its important roles in cell physiology and success in targeted analysis of individual substrates, system-wide characterization and functional quantification of the pathway have been hindered by the lack effective tools and strategies for global site-specific identification of proline hydroxylation targets. Our overall hypothesis and long-term goal is that systematic characterization of ?proline hydroxylome? through the development of functional proteomics approaches will lead to mechanistic understanding of the novel Hyp-mediated metabolic regulations in development and diseases. To achieve this goal, we have developed and applied an immunoprecipitation-assisted strategy for global identification of proline hydroxylation targets. With this strategy, we will tackle the challenge of systematic discovery and quantification of proline hydroxylation proteome. We will develop new quantitative proteomics workflows and apply the strategies for the identification and validation of endogenous prolyl hydroxylase targets. Using temporal dynamics analysis, we will also reveal the target proteins that are subject to Hyp-dependent protein degradation. Integrated data analysis will reveal the regulatory enzyme of the novel Hyp substrates and therefore enable confident validation as well as functional characterization. In addition to the target-specific degradation, our preliminary proteomics analysis showed that proline hydroxylation may regulate global protein homeostasis through the regulation of proteasome activities. We will develop endogenous model systems and novel quantitative mass spectrometry technology to determine the functional significance of proline hydroxylation on proteasome subunits and how such regulation affect global protein homeostasis. Overall, we anticipate that the development and application of functional proteomics technology for system-wide analysis of proline hydroxylation targets will reveal novel metabolic-sensing pathways and potentially lead to paradigm- shifting concepts in the fields of cancer, metabolic diseases and aging.